Method of modulating endothelial cell activity

ABSTRACT

The present invention relates generally to a method of modulating endothelial cell activity and to agents useful for same. More particularly, the present invention relates to a method of modulating intercellular vascular endothelial permeability by modulating an intracellular protein kinase C-dependent signalling mechanism. The method for the present invention is useful, inter alia, in the treatment and/or prophylaxis of a condition charicterised by aberrant, unwanted or otherwise inappropriate endothelial cell activity, in particular, conditions characterised by a loss of vascular intergrity.

FIELD OF THE INVENTION

The present invention relates generally to a method of modulatingendothelial cell activity and to agents useful for same. Moreparticularly, the present invention relates to a method of modulatingintercellular vascular endothelial permeability by modulating anintracellular protein kinase C-dependent signalling mechanism. Themethod for the present invention is useful, inter alia, in the treatmentand/or prophylaxis of a condition characterised by aberrant, unwanted orotherwise inappropriate endothelial cell activity, in particular,conditions characterised by a loss of vascular integrity.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected alphabetically at the end of thedescription.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in Australia or anyother country.

One of the essential functions of the endothelial cell lining is tomaintain the essentially impermeable nature of the blood vesselcontrolling the passage of solutes and inflammatory cells from thecirculation to the tissues. Endothelial cell hyper-permeability is acharacteristic of blood vessels in many pathologies. For example, newlyformed micro-vessels in tumours are highly permeable. Indeed, suchhyper-permeability allows the deposition of fibrin in tumours thatsupports and promotes cell adhesion and migration, essential steps inthe angiogenic response (Dvorak, H. F., Harvey, V. S., Estrella, P.,Brown, L. F., McDonagh, J., Dvorak, A. M. (1987) Lab Invest. 57:673-86;Dvorak, H. F., Brown, L. F., Detmar, M., Dvorak, A. M. (1995) Am JPathol. 146:1029-39). In chronic inflammatory states such as inrheumatoid arthritis and atherosclerosis, vessel hyper-permeabilityallows increased transmigration of inflammatory cells across theactivated endothelium. A number of factors have previously beendescribed which promote endothelial cell leakiness, for example,thrombin, tumour necrosis-factor and vascular endothelial cell growthfactor (VEGF). These appear to act by inducing changes in junctionalmolecules such as PECAM-1 and VE-cadherin or their associated signallingmolecules, such as the catenins.

Thrombin is a serine protease with multiple roles central to vascularbiology, acting upon platelets, endothelial cells and circulatingclotting factors (Macfarlane, S. R., Seatter, M. J., Kantce, T., Hunter,G. D., Plevin, R. (2001) Pharmacol Rev. 53:245-82). Thrombin is a potentactivator of endothelial cells, increasing intercellular gap formationand the permeability of confluent endothelial cell monolayers (Lum, H.,Andersen, T. T., Siflinger-Birnboim, A., Tiruppathi, C., Goligorsky, M.S., Fenton, J. W. 2nd, Malik, A. B. (1993) J Cell Biol. 120:1491-9;Garcia, J. G., Verin, A. D., Schaphorst, K. L. (1996) Semin ThrombHemost. 22:309-15). Thrombin signaling is mediated by theprotease-activated receptor PAR-1 (Coughlin et al. (2000) supra).Thrombin cleaves the PAR-1 ligand from the receptor thus allowing theligand to activate receptor signaling. PAR-1 can activate a number ofdownstream signaling pathways, the molecules activated depend upon theG-proteins recruited to the receptor (Maefarlane et al. (2001) supra).

The protein kinase Cζ family of serine/threonine kinases are involved insignal transduction and are dependent upon lipids for their activity.The isoforms of protein kinase Cζ are classified according to theirstructure and activation/substrate requirements (Draijer, R., Atsma, D.E., van der Laarse, A., van Hinsbergh, V. W. (1995) Circ Res.76:199-208). The classical protein kinase Cζs (α, βI, βII, γ) areCa²⁺-dependent and regulated by diacyglycerol (DAG) orphosphotidylserine (PS), the novel protein kinase Cζs (δ, ε, η, θ) arealso activated by DAG or PS but are Ca²⁺-independent, while the atypicalprotein kinase Cζs (ζ, 1/λ) are regulated by PS, independent of both DAGand Ca²⁺. ¹⁰ The activity of protein kinase Cζs is controlled by theirphosphorylation status and relocalisation (Parelk, D. B., Ziegler, W.,Parker, P. J. (2000) Embo J. 19:496-503). The biological consequences ofactivation of particular isoforms of protein kinase Cζ under specificconditions have not yet been determined.

Protein kinase C has been demonstrated to function as one such PAR-1downstream intermediate (Malik, 1994). However, studies of the effectsof protein kinase C activation on endothelial cell permeability haveproduced conflicting results with some authors concluding that proteinkinase C activation does not play a significant role in permeabilitychanges in endothelial cells (van Nieuw Amerongen G P, Draijer R,Vermeer M A, van Hinsbergh V W. (1998) Circ Res. 83:1115-23;Vouret-Craviari V, Boquet P, Pouyssegur J, Van Obberghen-Schilling E.(1998) Mol Biol Cell. 9:2639-53) while others conclude that proteinkinase C activation is important (Lynch J J, Ferro T J, Blumenstock F A,Brockenauer A M, Malik A B. (1990) J Clin Invest. 85:1991-8; Hempel,1997). Studies using phorbol-12-myristate-13-acetate as a diacylglycerolsurrogate to stimulate protein kinase C activation have shown bothinhibition (Yamada Y, Furumichi T, Furui H, Yokoi T, Ito T, Yamauchi K,Yokota M, Hayashi H, Saito H. (1990) Arteriosclerosis 10:410-20; vanNieuw Amerongen, 1998 supra) and stimulation (Lynch, 1990 supra;Bussolino F, Silvagno F, Garbarino G, Costamagna C, Sanavio F, Arese M,Soldi R, Aglietta M, Pescannona G, Camussi G, et al. (1994) J Biol Chem.269:2877-86; van Nieuw Amerongen, 1998 supra) of endothelial cellpermeability in a concentration dependent manner (Lynch, 1990 supra; vanNieuw Amerongen, 1998 supra). However, phorbol esters do not activateatypical protein kinase Cs (Zhou G, Wooten M W, Coleman E S. (1994) ExpCell Res. 214:1-11).

The signals which regulate endothelial cell permeability have clearlynot been fully defined. However, elucidation of these cellularsignalling mechanisms is essential for the development of therapeuticand/or prophylactic strategies directed to treating conditionscharacterised by aberrant or otherwise unwanted endothelial cellpermeability.

In work leading up to the present invention, it has been determined thatthe atypical protein kinase Cζ plays a functional role in the regulationof endothelial cell permeability. In particular, it has beensurprisingly determined that signalling relating to modulation ofintercellular endothelial cell permeability critically involvesactivation of this atypical form of protein kinase C, resulting interalia, in increased intercellular endothelial cell permeability. Sinceendothelial cell integrity is influenced by a balance between theinfluence of inflammatory mediators (such as thrombin) which increaseintercellular gap formation and promotes endothelial permeability andanti-inflammatory agents which promote cell cell junction formation andantagonise changes in endothelial cell permeability, the elucidation ofthis cellular signalling mechanism now facilitates the rational designof methodology directed to modulating endothelial cell activity byregulating the functioning of protein kinase Cζ.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

As used herein, the term “derived from” shall be taken to indicate thata specified integer may be obtained from a particular source albeit notnecessarily directly from that source.

One aspect of the present invention is directed to a method ofmodulating endothelial cell activity, said method comprising modulatingthe functional activity of protein kinase Cζ wherein up-regulatingprotein kinase Cζ activity to a functionally effective levelup-regulates said cellular activity and down-regulation protein kinaseCζ activity to a functionally ineffective level down-regulates saidcellular activity.

There is more particularly provided a method of modulating vascularendothelial cell activity, said method comprising modulating thefunctional activity of protein kinase Cζ wherein up-regulating proteinkinase Cζ activity to a functionally effective level up-regulates saidvascular endothelial activity and down-regulating protein kinase Cζactivity to a functionally ineffective level down-regulates saidvascular endothelial cell activity.

In another aspect there is provided a method of modulating intercellularvascular endothelial cell permeability, said method comprisingmodulating the functional activity of protein kinase Cζ whereinup-regulating protein kinase Cζ to a functionally effective levelup-regulates said intercellular vascular cell permeability anddown-regulating protein kinase Cζ activity to a functionally ineffectivelevel down-regulates said intercellular vascular endothelial cellpermeability.

Yet another aspect provides a method of modulating thrombin-inducedvascular endothelial cell activity, said method comprising modulatingthe functional activity of protein kinase Cζ wherein up-regulatingprotein kinase Cζ activity to a functionally effective levelup-regulates said vascular endothelial activity and down-regulatingprotein kinase Cζ activity to a functionally ineffective leveldown-regulates said vascular endothelial cell activity.

Still another aspect provides a method of modulating thrombin inducedintercellular vascular endothelial cell permeability, said methodcomprising modulating the functional activity of protein kinase Cζwherein up-regulating protein kinase Cζ activity to a functionallyeffective level up-regulates said permeability and down-regulating saidprotein kinase Cζ activity to a functionally ineffective leveldown-regulates said permeability.

Yet still another aspect of the present invention is directed to amethod of regulating endothelial cell activity in a mammal, said methodcomprising modulating the functional activity of protein kinase Cζ insaid mammal wherein up-regulating protein kinase Cζ activity to afunctionally effective level up-regulates said endothelial cell activityand down-regulating protein kinase Cζ activity to a functionallyineffective level down-regulates said endothelial cell activity.

In still yet another aspect there is provided a method of modulatingvascular endothelial cell activity in a mammal, said method comprisingmodulating the functional activity of protein kinase Cζ whereinup-regulating protein kinase Cζ activity to a functionally effectivelevel up-regulates said endothelial cell activity and down-regulatingprotein kinase Cζ activity to a functionally ineffective leveldown-regulates said endothelial cell activity.

In a further aspect there is provided a method of up-regulating vascularendothelial cell activity in a mammal, said method comprisingadministering to said mammal an effective amount of an agent for a timeand under conditions sufficient to induce a functionally effective levelof protein kinase Cζ.

In another further aspect there is provided a method of up-regulatingvascular endothelial cell activity in a mammal, said method comprisingadministering to said mammal an effective amount of protein kinase Cζfor a time and under conditions sufficient to induce a functionallyeffective level of protein kinase Cζ.

In still another further aspect there is provided a method ofup-regulating vascular endothelial cell activity in a mammal, saidmethod comprising administering to said mammal an effective amount of anucleotide sequence encoding protein kinase Cζ for a time and underconditions sufficient to induce a functionally effective level ofprotein kinase Cζ.

In yet still another aspect there is provided a method ofdown-regulating vascular endothelial cell activity in a mammal, saidmethod comprising administering to said mammal an effective amount of anagent for a time and under conditions sufficient to induce afunctionally ineffective level of protein kinase Cζ.

Another aspect of the present invention contemplates a method for thetreatment and/or prophylaxis of a condition characterised by aberrant,unwanted or otherwise inappropriate endothelial cell activity in amammal, said method comprising modulating the functional activity ofprotein kinase Cζ wherein up-regulating protein kinase Cζ activity to afunctionally effective level up-regulates said endothelial cell activityand down-regulating protein kinase Cζ activity to a functionallyineffective level down-regulates said cellular activity.

In yet another aspect there is provided a method for the treatmentand/or prophylaxis of a condition characterised by unwanted vascularendothelial cell activity in a mammal, said method comprisingadministering to said mammal an effective amount of an agent for a timeand under conditions sufficient to induce a functionally ineffectivelevel of protein kinase Cζ.

Still another aspect of the present invention relates to the use of anagent capable of modulating the functionally effective level of proteinkinase Cζ in the manufacture of a medicament for the regulation ofthrombin-induced endothelial cell activity in a mammal whereinup-regulating protein kinase Cζ activity to a functionally effectivelevel up-regulates said endothelial cell activity and down-regulatingprotein kinase Cζ activity to a functionally ineffective leveldown-regulates said endothelial cell activity.

In another aspect the present invention relates to the use of proteinkinase Cζ or a nucleic acid encoding protein kinase Cζ in themanufacture of a medicament for the regulation of endothelial cellactivity wherein up-regulating protein kinase Cζ to a functionally levelup-regulates said endothelial cell activity.

In yet another further aspect, the present invention contemplates apharmaceutical composition comprising the modulatory agent ashereinbefore defined and one or more pharmaceutically acceptablecarriers and/or diluents. Said agents are referred to as the activeingredients

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of protein kinase C inhibitorsselectively blocking thrombin stimulated permeability increases. Cellswere untreated (Nil), treated with thrombin (0.2 U/ml), (T) for 15 mins,pre-treated with inhibitor for 15 mins or pre-treated with inhibitorfollowed by thrombin. a, Bisindolylmaleimide I blocksthrombin-stimulated permeability increases in endothelial cells at highconcentrations (BisLo, 100 nM; BisHi, 6 μM). b, Chelerythrine chlorideblocks thrombin-stimulated permeability increases in endothelial cells(Cζ, 1 μm). c, The specific PKA inhibitor H-89 has no effect onthrombin-stimulated permeability increases in endothelial cells (H-89,50 nM).d, Calphostin C does not block thrombin-stimulated permeabilityincreases in endothelial cells (CalC, 100 nM). Relative fluorescentemission at 530 nm is shown. Data are mean ±SEM of duplicatedeterminations in each group and are representative of at least 3independent experiments.

FIG. 2 is an image of the treatment of endothelial cells with thrombininducing protein kinase Cζ relocalisation. Endothelial cell monolayerswere stained to demonstrate changes in protein kinase Cζ (a-c)andprotein kinase Cζ (d-f) localization following treatment with thrombin(0.2 U/ml). a, control antibody. b, untreated. c, post thrombintreatment. d, control antibody. e, untreated. f, post thrombintreatment.

FIG. 3 a is a graphical representation of dominant-negative proteinkinase Cζ blocks thrombin stimulated permeability increases inendothelial cells. Endothelial cell were infected with pAdEasy-1adenovirus empty vector (EV) or pAdEasy-1 constructs encodingdominant-negative protein kinase Cζ (DN) or constitutively activeprotein kinase Cζ (CA) prior to assay. Cells were either untreated (−)or treated with thrombin at 0.2 U/ml (+). Relative fluorescent emissionat 530 nm is shown. Data shown are mean ±SEM of triplicatedeterminations and are representative of 3 independent experiments.

FIG. 3 b is a graphical representation of angiopoietin-1 inhibitingthrombin stimulated endothelial cell permeability increases. Cells wereuntreated (Nil), treated with angiopoietin-1 (0.1 μl/ml) (Ang-1) for 30min, treated with thrombin (0.2 U/ml), (Thrombin) for 15 mins,pre-treated with angiopoietin-1 (0.1 μg/ml) for 30 min followed bythrombin (Ang-1+T). Relative fluorescent emission at 530 nm is shown.Data shown are mean ±SEM of triplicate determinations and arerepresentative of 3 independent experiments.

FIG. 4 is an image of angiopoietin-1 blocking thrombin stimulatedprotein kinase Cζ relocalisation. Endothelial cell monolayers werestained to demonstrate changes in protein kinase Cζ localisationfollowing treatment with thrombin (0.2 U/ml) either alone or afterpre-treatment with angiopoietin-1 (0.1 μg/ml), the pan-protein kinase Cinhibitor bisindolylmaleimide I (6 μM) or the PI3-kinase inhibitorLY294002 (10 μM). a, No treatment. b, Thrombin alone. c, angiopoietin-1and thrombin. d, bisindolylmaleimide I and thrombin. e, LY294002 andthrombin.

FIG. 5 is an image of angiopoietin-1 blocking thrombin stimulatedprotein kinase Cζ phoshorylation. a) Endothelial cells were infectedwith FLAG-protein kinase Cζ in the pAdEasy-1 adenovirus vector. Cellswere lysed after treatment with thrombin (0.2 U/ml) either alone orafter pre-treatment with angiopoietin-1 (0.1 μg/ml), the pan-proteinkinase C inhibitor bisindolylmaleimide 1 (6 μM) or the PI3-kinaseinhibitor LY294002 (10 μM). Proteins were separated by SDS-PAGE beforetransfer to PVDF membrane and probed with an antibody directed againstphosphorylated Thr410 of the activation loop of protein kinase Cζ (upperpanel). Membranes were stripped and re-probed with an anti-proteinkinase Cζ antibody (lower panel). Nil, No treatment. T, Thrombin alone.Ang-1+T, angiopoietin-1 and thrombin. Bis+T, bisindolylmaleimide I andthrombin. LY+T, LY294002 and thrombin. b) Endothelial cells infectedwith pAdEasy-1 constructs encoding dominant-negative protein kinase Cζor constitutively active protein kinase Cζ. Cells were either untreatedor treated with thrombin, lysed and processed as for 5a. DN,dominant-negative protein kinase Cζ over-expressing cells. DN+T,dominant-negative protein kinase Cζ over-expressing cells and thrombintreated. CA, constitutively active protein kinase Cζ over-expressingcells. CA+T, constitutively active protein kinase Cζ over-expressingcells and thrombin treated.

FIG. 6 is a graphical representation of the PI3-kinase inhibitorLY294002 blocking thrombin-stimulated permeability increases inendothelial cells. Cells were untreated (Nil), treated with thrombin(0.2 U/ml), (T) for 15 mins, pre-treated with LY294002 (10 μM) (LY) for15 mins or pre-treated with LY294002 followed by thrombin (LY+T). (+).Relative fluorescent emission at 530 nm is shown. Data shown are mean±SEM of triplicate determinations and are representative of 3independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the determination thatthe endothelial cell signalling related to modulation of intercellularpermeability critically involves activation of the atypical proteinkinase Cζ. This development now permits the rational design oftherapeutic and/or prophylactic methods for treating conditionscharacterised by aberrant or unwanted endothelial cell activity.

Accordingly, one aspect of the present invention is directed to a methodof modulating endothelial cell activity, said method comprisingmodulating the functional activity of protein kinase Cζ whereinup-regulating protein kinase Cζ activity to a functionally effectivelevel up-regulates said cellular activity and down-regulation proteinkinase Cζ activity to a functionally ineffective level down-regulatessaid cellular activity.

Reference to “endothelial cell” should be understood as a reference tothe cells which line the blood vessels, lymphatics or other serouscavities such as fluid filled cavities. The phrase “endothelial cells”should also be understood as a reference to cells which exhibit one ormore of the morphology, phenotype and/or functional activity ofendothelial cells and is also a reference to mutants or variantsthereof. “Variants” include, but are not limited to, cells exhibitingsome but not all of the morphological or phenotypic features orfunctional activities of endothelial cells at any differentiative stageof development. “Mutants” include, but are not limited to, endothelialcells which have been naturally or non-naturally modified such as cellswhich are genetically modified.

It should also be understood that the endothelial cells of the presentinvention may be at any differentiative stage of development.Accordingly, the cells may be immature and therefore functionallyincompetent in the absence of further differentiation. In this regard,highly immature cells such as stem cells, which retain the capacity todifferentiate into endothelial cells, should nevertheless be understoodto satisfy the definition of “endothelial cell” as utilised herein dueto their capacity to differentiate into endothelial cells underappropriate conditions. Preferably, the subject endothelial cell is avascular endothelial cell or a lymphatic endothelial cell.

Accordingly, there is more particularly provided a method of modulatingvascular endothelial cell activity, said method comprising modulatingthe functional activity of protein kinase Cζ wherein up-regulatingprotein kinase Cζ activity to a functionally effective levelup-regulates said vascular endothelial activity and down-regulatingprotein kinase Cζ activity to a functionally ineffective leveldown-regulates said vascular endothelial cell activity.

There is also provided a method of modulating lymphatic endothelial cellactivity, said method comprising modulating the functional activity ofprotein kinase Cζ wherein up-regulating protein kinase Cζ activity to afunctionally effective level up-regulates said lymphatic endothelialactivity and down-regulating protein kinase Cζ activity to afunctionally ineffective level down-regulates said lymphatic endothelialcell activity.

Reference to endothelial cell “activity” should be understood as areference to any one or more of the functional activities which anendothelial cell is capable of performing, for example, as a result ofstimulation by an extracellular agent such as thrombin, VEGF or TNF.Preferably, said activity is endothelial cell permeability.

Without limiting the present invention to any one theory or mode ofaction, it is thought that modulation of endothelial cell permeabilitycan occur at the level of modulating intercellular permeability and/orintracellular permeability. It should be understood that in a preferredembodiment, the present invention is directed to modulation of one orboth of these mechanisms of permeability.

According to this preferred embodiment there is provided a method ofmodulating intercellular endothelial cell permeability, said methodcomprising modulating the functional activity of protein kinase Cζwherein up-regulating protein kinase Cζ to a functionally effectivelevel up-regulates said intercellular cell permeability anddown-regulating protein kinase Cζ activity to a functionally ineffectivelevel down-regulates said intercellular endothelial cell permeability.

In another preferred embodiment there is provided a method of modulatingintracellular endothelial cell permeability, said method comprisingmodulating the functional activity of protein kinase Cζ whereinup-regulating protein kinase Cζ to a functionally effective levelup-regulates said intracellular cell permeability and down-regulatingprotein kinase Cζ activity to a functionally ineffective leveldown-regulates said intracellular endothelial cell permeability.

Still more preferably said endothelial cell is a vascular endothelialcell or a lymphatic endothelial cell.

Reference to “protein kinase Cζ” should be understood as a reference toall forms of this protein and to functional derivatives, homologues,analogues, chemical equivalents or mimetics thereof. This includes, forexample, any isoforms which arise from alternative splicing of thesubject protein kinase Cζ mRNA or mutants or polymorphic variants ofthese proteins.

Without limiting the present invention to any one theory or mode ofaction, thrombin signalling leads to, inter alia, increasedintercellular vascular endothelial cell permeability. However, since thethrombin receptor PAR-1 activates a diverse range of downstreamsignalling intermediates, the precise signalling pathway involved inthis mechanism had previously been obscure. It has now been determinedthat the atypical form of protein kinase C, being the protein kinase Cζisoform, regulates vascular endothelial cell leakiness via thePI3-kinase dependent pathway. However, although the present inventionhas been exemplified with respect to thrombin, this should not beunderstood as a limitation with respect to the present invention whichnevertheless encompasses modulation of endothelial cell permeability byany means including, but not limited to, via thrombin, VEGF and TNFstimulation. It has been determined that endothelial cell permeability,per se, is regulated via the protein kinase Cζ signalling pathway,irrespective of the nature of the specific stimulatory or inhibitorysignal.

Reference to “modulating” should be understood as a reference toup-regulating or down-regulating the subject endothelial cell activity.Reference to “down-regulating” endothelial cell activity shouldtherefore be understood as a reference to preventing, reducing (eg.slowing) or otherwise inhibiting one or more aspects of the functioningof the endothelial cell (for example retarding or preventingthrombin-induced vascular endothelial cell intercellular permeability)while reference to “up-regulating” should be understood to have theconverse meaning.

Still without limiting the present invention in any way, thrombin is ahighly pleiotropic molecule which exhibits multiple roles central tovascular biology including, but not limited to, acting upon platelets,endothelial cells and circulating clotting factors. Thrombin is a potentactivator of vascular endothelial cells, increasing intercellular gapformation and permeability of confluent vascular endothelial cellmonolayers. It is thought that the protein kinase Cζ mediated thrombinactivity predominantly relates to increasing endothelial cellpermeability (either intercellularly or intracellularly) and thereforeregulating endothelial cell leakiness. Although the present inventionextends to modulation of vascular permeability, per se, in a preferredembodiment, said vascular permeability is thrombin-induced permeability.

The present invention therefore most preferably provides a method ofmodulating thrombin induced intercellular endothelial cell permeability,said method comprising modulating the functional activity of proteinkinase Cζ wherein up-regulating protein kinase Cζ activity to afunctionally effective level up-regulates said permeability anddown-regulating said protein kinase Cζ activity to a functionallyineffective level down-regulates said permeability.

More preferably, said endothelial cell is a vascular endothelial cell ora lymphatic endothelial cell.

Even more preferably, said permeability is either intercellular orintracellular.

By “thrombin” is meant all forms of thrombin and functional derivatives,homologues, analogues, chemical equivalents and mimetics thereof.Reference to “thrombin” should also be understood to include referenceto any isoforms which arise from alternative splicing of thrombin mRNAor mutants or polymorphic variants of thrombin. It should also beunderstood to include reference to any other molecule which exhibitsthrombin functional activity to the extent that the subject moleculemimics one or more thrombin signalling events by inducing signallingthrough a thrombin or thrombin-like receptor. Without limiting thepresent invention to any one theory or mode of action, thrombin signalsvia the protease activated receptor PAR-1. Protein kinase Cζ isactivated via the PI3 kinase dependent pathway to induce vascularleakage. Since the method of the present invention is directed tomodulating endothelial cell activity by modulating an intracellularsignalling event which has been induced as a result of the interactionof thrombin with its receptor, this methodology can be applied tomodulating such a cellular activity, irrespective of whether it has beeninduced by the interaction of thrombin with a PAR-1 receptor or theinteraction of a thrombin mimetic, such as the naturally occurring ornon-naturally occurring mimetic or analogue, with the subject receptor.It is conceivable, for example, that there may be naturally ornon-naturally occurring thrombin mimetics (for example, toxins or drugs)which, if they were introduced into an individual, would induce unwantedthrombin-like endothelial cell activities due to their interaction withthe PAR-1 receptor. Accordingly, the present invention should beunderstood to extend to the modulation of such cellular activities whichare herein defined as falling within the scope of being “thrombininduced”. It should also be understood that to the extent that it may bedesirable to up-regulate the cellular activity which is normally inducedby thrombin, the method of the present invention provides a means ofoptionally circumventing the requirement for thrombin stimulation bydirectly up-regulating the protein kinase Cζ intracellular signallingpathway. It should be understood that this type of up-regulationnevertheless falls within the scope of a “thrombin-induced” activitysince the method of the present invention effectively provides a meansof mimicking this type of activity.

Reference to protein kinase Cζ “activity” should be understood as areference to any one or more of the activities which protein kinase Cζcan perform. For example, and without limiting the present invention inany way, the activity of protein kinase Cζ is controlled by itsphosphorylation status and relocalisation. Specifically, protein kinaseCζ is one of the substrates for phosphoinositide-dependent kinase-1(PDK-1), a pivotal component of the phosphionositide 3 OH-kinase(PI3-kinase) signalling pathway. Subsequently to protein kinase Cζactivation, downstream signalling events include alteration ofjunctional molecules such as PECAM, VE cadherin, JAM-2 and catenins,which are all involved with regulation of junctional integrity inendothelial cells. Protein kinase Cζ is also thought to affect the actincytoskeleton through modulation of Rho and rac. Accordingly, referenceto “modulating” protein kinase Cζ functional activity is a reference toeither up-regulating or down-regulating protein kinase Cζ functionalactivity. Such modulation may be achieved by any suitable means andincludes:

-   -   (i) Modulating absolute levels of the active or inactive forms        of protein kinase Cζ (for example increasing or decreasing        intracellular protein kinase Cζ concentrations) such that either        more or less protein kinase Cζ is available for activation        and/or to interact with its downstream targets.    -   (ii) Agonising or antagonising protein kinase Cζ such that the        functional effectiveness of any given protein kinase Cζ molecule        is either increased, decreased or otherwise modulated or        changed. For example, increasing the half life of protein kinase        Cζ may achieve an increase in the overall level of protein        kinase Cζ activity without actually necessitating an increase in        the absolute intracellular concentration of protein kinase Cζ.        Similarly, the partial antagonism of protein kinase Cζ, for        example by coupling protein kinase Cζ to a molecule that        introduces some steric hindrance in relation to the binding of        protein kinase Cζ to its downstream targets, may act to reduce,        although not necessarily eliminate, the effectiveness of protein        kinase Cζ signalling. Accordingly, this may provide a means of        down-regulating protein kinase Cζ functioning without        necessarily down-regulating absolute concentrations of protein        kinase Cζ.

In terms of achieving the up or down-regulation of protein kinase Cζfunctioning, means for achieving this objective would be well known tothe person of skill in the art and include, but are not limited to:

-   -   (i) Introducing into a cell a nucleic acid molecule encoding        protein kinase Cζ or functional equivalent, derivative or        analogue thereof in order to up-regulate the capacity of said        cell to express protein kinase Cζ.    -   (ii) Introducing into a cell a proteinaceous or        non-proteinaceous molecule which modulates transcriptional        and/or translational regulation of a gene, wherein this gene may        be a protein kinase Cζ gene or functional portion thereof or        some other gene which directly or indirectly modulates the        expression of the protein kinase Cζ gene.    -   (iii) Introducing into a cell the protein kinase Cζ expression        product (in either active or inactive form) or a functional        derivative, homologue, analogue, equivalent or mimetic thereof.    -   (iv) Introducing a proteinaceous or non-proteinaceous molecule        which functions as an antagonist to the protein kinase Cζ        expression product.    -   (v) Introducing a proteinaceous or non-proteinaceous molecule        which functions as an agonist of the protein kinase Cζ        expression product.    -   (vi) Introducing a proteinaceous or non-proteinaceous molecule        which modulates the nature of the functional activity exhibited        by protein kinase Cζ.

The proteinaceous molecules described above may be derived from anysuitable source such as natural, recombinant or synthetic sources andincludes fusion proteins or molecules which have been identifiedfollowing, for example, natural product screening. The reference tonon-proteinaceous molecules may be, for example, a reference to anucleic acid molecule or it may be a molecule derived from naturalsources, such as for example natural product screening, or may be achemically synthesised molecule. The present invention contemplatesanalogues of the protein kinase Cζ expression product or small moleculescapable of acting as agonists or antagonists. Chemical agonists may notnecessarily be derived from the protein kinase Cζ expression product butmay share certain conformational similarities. Alternatively, chemicalagonists may be specifically designed to meet certain physiochemicalproperties. Antagonists may be any compound capable of blocking,inhibiting or otherwise preventing protein kinase Cζ from carrying outits normal biological function, such as molecules which prevent itsactivation or else prevent the downstream functioning of activatedprotein kinase Cζ. Antagonists include monoclonal antibodies andantisense nucleic acids which prevent transcription or translation ofprotein kinase Cζ genes or mRNA in mammalian cells. Modulation ofexpression may also be achieved utilising antigens, RNA, ribosomes,DNAzymes, RNA aptamers, antibodies or molecules suitable for use incosuppression. The proteinaceous and non-proteinaceous moleculesreferred to in points (i)-(v), above, are herein collectively referredto as “modulatory agents”.

Screening for the modulatory agents hereinbefore defined can be achievedby any one of several suitable methods including, but in no way limitedto, contacting a cell comprising the protein kinase Cζ gene orfunctional equivalent or derivative thereof with an agent and screeningfor the modulation of protein kinase Cζ protein production or functionalactivity, modulation of the expression of a nucleic acid moleculeencoding protein kinase Cζ or modulation of the activity or expressionof a downstream protein kinase Cζ cellular target. Detecting suchmodulation can be achieved utilising techniques such as Westernblotting, electrophoretic mobility shift assays and/or the readout ofreporters of protein kinase Cζ activity such as luciferases, CAT and thelike.

It should be understood that the protein kinase Cζ gene or functionalequivalent or derivative thereof may be naturally occurring in the cellwhich is the subject of testing or it may have been transfected into ahost cell for the purpose of testing. Further, the naturally occurringor transfected gene may be constitutively expressed—thereby providing amodel useful for, inter alia, screening for agents which down regulateprotein kinase Cζ activity, at either the nucleic acid or expressionproduct levels, or the gene may require activation—thereby providing amodel useful for, inter alia, screening for agents which up regulateprotein kinase Cζ expression. Further, to the extent that a proteinkinase Cζ nucleic acid molecule is transfected into a cell, thatmolecule may comprise the entire protein kinase Cζ gene or it may merelycomprise a portion of the gene such as the portion which regulatesexpression of the protein kinase Cζ product. For example, the proteinkinase Cζ promoter region may be transfected into the cell which is thesubject of testing. In this regard, where only the promoter is utilised,detecting modulation of the activity of the promoter can be achieved,for example, by ligating the promoter to a reporter gene. For example,the promoter may be ligated to luciferase or a CAT reporter, themodulation of expression of which gene can be detected via modulation offluorescence intensity or CAT reporter activity, respectively.

In another example, the subject of detection could be a downstreamprotein kinase Cζ regulatory target, rather than protein kinase Cζitself. Yet another example includes protein kinase Cζ binding sitesligated to a minimal reporter. For example, modulation of protein kinaseCζ activity can be detected by screening for the modulation of thefunctional activity in an endothelial cell. This is an example of anindirect system where modulation of protein kinase Cζ expression, perse, is not the subject of detection. Rather, modulation of the moleculeswhich protein kinase Cζ regulates the expression of, are monitored.

These methods provide a mechanism for performing high throughputscreening of putative modulatory agents such as the proteinaceous ornon-proteinaceous agents comprising synthetic, combinatorial, chemicaland natural libraries. These methods will also facilitate the detectionof agents which bind either the protein kinase Cζ nucleic acid moleculeor expression product itself or which modulate the expression of anupstream molecule, which upstream molecule subsequently modulatesprotein kinase Cζ expression or expression product activity.Accordingly, these methods provide a mechanism of detecting agents whicheither directly or indirectly modulate protein kinase Cζ expressionand/or activity.

It should be understood that a related aspect of the present inventionis directed to methods of screening for said modulatory agents.

The agents which are utilised in accordance with the method of thepresent invention may take any suitable form. For example, proteinaceousagents may be glycosylated or unglycosylated, phosphorylated ordephosphorylated to various degrees and/or may contain a range of othermolecules used, linked, bound or otherwise associated with the proteinssuch as amino acids, lipid, carbohydrates or other peptides,polypeptides or proteins. Similarly, the subject non-proteinaceousmolecules may also take any suitable form. Both the proteinaceous andnon-proteinaceous agents herein described may be linked, bound otherwiseassociated with any other proteinaceous or non-proteinaceous molecules.For example, in one embodiment of the present invention, said agent isassociated with a molecule which permits its targeting to a localisedregion.

The subject proteinaceous or non-proteinaceous molecule may act eitherdirectly or indirectly to modulate the expression of protein kinase Cζor the activity of the protein kinase Cζ expression product. Saidmolecule acts directly if it associates with the protein kinase Cζnucleic acid molecule or expression product to modulate expression oractivity, respectively. Said molecule acts indirectly if it associateswith a molecule other than the protein kinase Cζ nucleic acid moleculeor expression product which other molecule either directly or indirectlymodulates the expression or activity of the protein kinase Cζ nucleicacid molecule or expression product, respectively. Accordingly, themethod of the present invention encompasses the regulation of proteinkinase Cζ nucleic acid molecule expression or expression productactivity via the induction of a cascade of regulatory steps.

The term “expression” refers to the transcription and translation of anucleic acid molecule. Reference to “expression product” is a referenceto the product produced from the transcription and translation of anucleic acid molecule. Reference to “modulation” should be understood asa reference to up-regulation or down-regulation.

“Derivatives” of the molecules herein described (for example thrombin,protein kinase Cζ or other proteinaceous or non-proteinaceous agents)include fragments, parts, portions or variants from either natural ornon-natural sources. Non-natural sources include, for example,recombinant or synthetic sources. By “recombinant sources” is meant thatthe cellular source from which the subject molecule is harvested hasbeen genetically altered. This may occur, for example, in order toincrease or otherwise enhance the rate and volume of production by thatparticular cellular source. Parts or fragments include, for example,active regions of the molecule. Derivatives may be derived frominsertion, deletion or substitution of amino acids. Amino acidinsertional derivatives include amino and/or carboxylic terminal fusionsas well as intrasequence insertions of single or multiple amino acids.Insertional amino acid sequence variants are those in which one or moreamino acid residues are introduced into a predetermined site in theprotein although random insertion is also possible with suitablescreening of the resulting product. Deletional variants arecharacterised by the removal of one or more amino acids from thesequence. Substitutional amino acid variants are those in which at leastone residue in a sequence has been removed and a different residueinserted in its place. Additions to amino acid sequences include fusionswith other peptides, polypeptides or proteins, as detailed above.

Derivatives also include fragments having particular epitopes or partsof the entire protein fused to peptides, polypeptides or otherproteinaceous or non-proteinaceous molecules. For example, proteinkinase Cζ or derivative thereof may be fused to a molecule to facilitateits entry into a cell. Analogs of the molecules contemplated hereininclude, but are not limited to, modification to side chains,incorporating of unnatural amino acids and/or their derivatives duringpeptide, polypeptide or protein synthesis and the use of crosslinkersand other methods which impose conformational constraints on theproteinaceous molecules or their analogs.

Derivatives of nucleic acid sequences which may be utilised inaccordance with the method of the present invention may similarly bederived from single or multiple nucleotide substitutions, deletionsand/or additions including fusion with other nucleic acid molecules. Thederivatives of the nucleic acid molecules utilised in the presentinvention include oligonucleotides, PCR primers, antisense molecules,molecules suitable for use in cosuppression and fusion of nucleic acidmolecules. Derivatives of nucleic acid sequences also include degeneratevariants.

A “variant” or “mutant” of thrombin or protein kinase Cζ should beunderstood to mean molecules which exhibit at least some of thefunctional activity of the form of thrombin or protein kinase Cζ ofwhich it is a variant or mutant. A variation or mutation may tale anyform and may be naturally or non-naturally occurring.

A “homologue” is meant that the molecule is derived from a species otherthan that which is being treated in accordance with the method of thepresent invention. This may occur, for example, where it is determinedthat a species other than that which is being treated produces a form ofprotein kinase Cζ which exhibits similar and suitable functionalcharacteristics to that of the protein kinase Cζ which is naturallyproduced by the subject undergoing treatment.

Chemical and functional equivalents should be understood as moleculesexhibiting any one or more of the functional activities of the subjectmolecule, which functional equivalents may be derived from any sourcesuch as being chemically synthesised or identified via screeningprocesses such as natural product screening. For example chemical orfunctional equivalents can be designed and/or identified utilising wellknown methods such as combinatorial chemistry or high throughputscreening of recombinant libraries or following natural productscreening.

For example, libraries containing small organic molecules may bescreened, wherein organic molecules having a large number of specificparent group substitutions are used. A general synthetic scheme mayfollow published methods (eg., Bunin B A, et al. (1994) Proc. Natl. AcadSci. USA, 91:4708-4712; DeWitt S H, et al. (1993) Proc. Natl. Acad Sci.USA, 90:6909-6913). Briefly, at each successive synthetic step, one of aplurality of different selected substituents is added to each of aselected subset of tubes in an array, with the selection of tube subsetsbeing such as to generate all possible permutation of the differentsubstituents employed in producing the library. One suitable permutationstrategy is outlined in U.S. Pat. No. 5,763,263.

There is currently widespread interest in using combinational librariesof random organic molecules to search for biologically active compounds(see for example U.S. Pat. No. 5,763,263). Ligands discovered byscreening libraries of this type may be useful in mimicking or blockingnatural ligands or interfering with the naturally occurring ligands of abiological target. In the present context, for example, they may be usedas a starting point for developing protein kinase Cζ analogues whichexhibit properties such as more potent pharmacological effects. Proteinkinase Cζ or a functional part thereof may according to the presentinvention be used in combination libraries formed by various solid-phaseor solution-phase synthetic methods (see for example U.S. Pat. No.5,763,263 and references cited therein). By use of techniques, such asthat disclosed in U.S. Pat. No. 5,753,187, millions of new chemicaland/or biological compounds may be routinely screened in less than a fewweeks. Of the large number of compounds identified, only thoseexhibiting appropriate biological activity are further analysed.

With respect to high throughput library screening methods, oligomeric orsmall-molecule library compounds capable of interacting specificallywith a selected biological agent, such as a biomolecule, a macromoleculecomplex, or cell, are screened utilising a combinational library devicewhich is easily chosen by the person of skill in the art from the rangeof well-known methods, such as those described above. In such a method,each member of the library is screened for its ability to interactspecifically with the selected agent. In practising the method, abiological agent is drawn into compound-containing tubes and allowed tointeract with the individual library compound in each tube. Theinteraction is designed to produce a detectable signal that can be usedto monitor the presence of the desired interaction. Preferably, thebiological agent is present in an aqueous solution and furtherconditions are adapted depending on the desired interaction. Detectionmay be performed for example by any well-known functional ornon-functional based method for the detection of substances.

In addition to screening for molecules which mimic the activity ofprotein kinase Cζ, it may also be desirable to identify and utilisemolecules which function agonistically or antagonistically to proteinkinase Cζ in order to up or down-regulate the functional activity ofprotein kinase Cζ in relation to modulating endothelial cell growth. Theuse of such molecules is described in more detail below. To the extentthat the subject molecule is proteinaceous, it may be derived, forexample, from natural or recombinant sources including fusion proteinsor following, for example, the screening methods described above. Thenon-proteinaceous molecule may be, for example, a chemical or syntheticmolecule which has also been identified or generated in accordance withthe methodology identified above. Accordingly, the present inventioncontemplates the use of chemical analogues of protein kinase Cζ capableof acting as agonists or antagonists. Chemical agonists may notnecessarily be derived from protein kinase Cζ but may share certainconformational similarities. Alternatively, chemical agonists may bespecifically designed to mimic certain physiochemical properties ofprotein kinase Cζ. Antagonists may be any compound capable of blocking,inhibiting or otherwise preventing protein kinase Cζ from carrying outits normal biological functions. Antagonists include monoclonalantibodies specific for protein kinase Cζ or parts of protein kinase Cζ.

Analogues of protein kinase Cζ or of protein kinase Cζ agonistic orantagonistic agents contemplated herein include, but are not limited to,modifications to side chains, incorporating unnatural amino acids and/orderivatives during peptide, polypeptide or protein synthesis and the useof crosslinkers and other methods which impose conformationalconstraints on the analogues. The specific form which such modificationscan take will depend on whether the subject molecule is proteinaceous ornon-proteinaceous. The nature and/or suitability of a particularmodification can be routinely determined by the person of skill in theart.

For example, examples of side chain modifications contemplated by thepresent invention include modifications of amino groups such as byreductive alkylation by reaction with an aldehyde followed by reductionwith NaBH4; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivatisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; perfornic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alklaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or allylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringprotein synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids. A list of unnaturalamino acids contemplated herein is shown in Table 1. TABLE 1Non-conventional Non-conventional amino acid Code amino acid Codeα-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrateMgabu L-N-methylarginine Nmarg aminocyclopropane- CproL-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmaspaminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- NorbL-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglucyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanineCpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine NmleuD-arginine Darg L-N-methyllysine Nmlys D-aspartic acid DaspL-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine NmnleD-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid DgluL-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine NmpheD-isoleucine Dile L-N-methylproline Nmpro D-leucine DleuL-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine NmthrD-methionine Dmet L-N-methyltryptophan Nmtrp D-ornithine DornL-N-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline NmvalD-proline Dpro L-N-methylethylglycine Nmetg D-serine DserL-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine NleD-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl--aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl-Nmbcethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilise 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety.

Reference herein to attaining either a “functionally effective level” or“functionally ineffective level” of protein kinase Cζ should beunderstood as a reference to attaining that level of functionally activeprotein kinase Cζ at which modulation of endothelial cell activity canbe achieved, whether that be upregulation or down-regulation. In thisregard, it is within the skill of the person of skill in the art todetermine, utilising routine procedures, the threshold level offunctionally active protein kinase Cζ expression above which endothelialcell activity can be upregulated and below which endothelial cellactivity is downregulated. For example, suitable for use in this regardis any method which regulates the phosphorylation status or the cellularlocalisation of protein kinase Cζ, as would any method which is based onthe alteration of RNA synthesis of protein kinase Cζ (for example,antisense constructs, DNAzymes or RNA could change the levels ofproteins). It should be understood that reference to an “effectivelevel” means the level necessary to at least partly attain the desiredresponse. The amount will vary depending on the health and physicalcondition of the cellular population and/or individual being treated,the taxonomic group of the cellular population and/or individual beingtreated, the degree of up or down-regulation which is desired, theformulation of the composition which is utilised, the assessment of themedical situation and other relevant factors. Accordingly, it isexpected that this level may vary between individual situations, therebyfalling in a broad range, which can be determined through routinetrials.

The method of the present invention contemplates the modulation ofendothelial cell functioning both in vitro and in vivo. Although thepreferred method is to treat an individual in vivo it shouldnevertheless be understood that it may be desirable that the method ofthe invention may be applied in an in vitro environment, for example toprovide an in vitro model of endothelial cell permeability analysis. Inanother example the application of the method of the present inventionin an in vitro environment may extend to providing a read out mechanismfor screening technologies such as those hereinbefore described. Thatis, molecules identified utilising these screening techniques can beassayed to observe the extent and/or nature of their functional effecton endothelial cells which have been functionally modulated according tothe method of the present invention.

Although the preferred method is to down-regulate, in particular,thrombin-induced vascular endothelial cell activity, thereby essentiallydown-regulating intercellular vascular endothelial cell permeability(for example in order to down-regulate the progression of aninflammatory response or the development of a tumour), it should beunderstood that there may also be circumstances in which it is desirableto up-regulate intercellular vascular endothelial cell permeability. Forexample, and without limiting the present invention in any way, it hasbeen observed that newly formed micro-vessels in tumours are highlypermeable. This hyper-permeability allows the deposition of fibrin intumours that supports and promotes adhesion and migration, beingessential steps in the angiogenic response. To the extent that thisoccurs in tumours, it is clearly an object of the present invention todown-regulate the instance of such vascular hyper-permeability. However,in the context of organ or limb transplantation, up-regulating vascularhyper-permeability in order to facilitate angiogenesis in the new organor limb may be highly desirable. In another example, an increase invascular hyper-permeability may be desirable in terms of facilitatingthe local or systemic delivery of a drug.

Accordingly, another aspect of the present invention is directed to amethod of regulating endothelial cell activity in a mammal, said methodcomprising modulating the functional activity of protein kinase Cζ insaid mammal wherein up-regulating protein kinase Cζ activity to afunctionally effective level up-regulates said endothelial cell activityand down-regulating protein kinase Cζ activity to a functionallyineffective level down-regulates said endothelial cell activity.

Preferably, said endothelial cell is a vascular endothelial cell or alymphatic endothelial cell and said activity is intercellularendothelial cell permeability or intracellular endothelial cellpermeability. Even more preferably, said activity is thrombin-inducedvascular endothelial cell permeability.

Modulation of said protein kinase Cζ functional activity is achieved viathe administration of protein kinase Cζ, a nucleic acid moleculeencoding protein kinase Cζ or an agent which effects modulation ofprotein kinase Cζ activity or protein kinase Cζ gene expression (hereincollectively referred to as “modulatory agents”). As detailedhereinbefore, it should be understood that the method of the presentinvention may be effected either with or without the presence ofthrombin. In particular, and as detailed hereinbefore, the determinationof the intracellular signalling mechanism which is utilised by thrombinin order to up-regulate endothelial cell activity now provides a meansof modulating said activity either as a consequence of thrombinstimulation or as a means of circumventing the requirement for thrombinstimulation (this latter outcome is particularly useful in terms of theup-regulation of intercellular vascular endothelial cell permeability inthe absence of thrombin stimulation).

Accordingly, in one preferred embodiment there is provided the method ofup-regulating endothelial cell activity in a mammal, said methodcomprising administering to said mammal an effective amount of an agentfor a time and under conditions sufficient to induce a functionallyeffective level of protein kinase Cζ.

In another preferred embodiment there is provided a method ofup-regulating endothelial cell activity in a mammal, said methodcomprising administering to said mammal an effective amount of proteinkinase Cζ for a time and under conditions sufficient to induce afunctionally effective level of protein kinase Cζ.

In still another preferred embodiment there is provided a method ofup-regulating endothelial cell activity in a mammal, said methodcomprising administering to said mammal an effective amount of anucleotide sequence encoding protein kinase Cζ for a time and underconditions sufficient to induce a functionally effective level ofprotein kinase Cζ.

In yet another preferred embodiment there is provided a method ofdown-regulating endothelial cell activity in a mammal, said methodcomprising administering to said mammal an effective amount of an agentfor a time and under conditions sufficient to induce a functionallyineffective level of protein kinase Cζ.

In accordance with these preferred embodiments of the present invention,said endothelial cell is preferably a vascular or lymphatic endothelialcell and said activity is preferably intercellular or intracellularendothelial cell permeability. Still more preferably, said activity isthrombin-induced vascular endothelial cell permeability.

Reference to “induce” should be understood as a reference to achievingthe desired protein kinase Cζ level, whether that be a functionallyeffective level or a functionally ineffective level. Said induction ismost likely to be achieved via the up-regulation or down-regulation ofprotein kinase Cζ functional activity, as hereinbefore described,although any other suitable means of achieving induction arenevertheless herewith encompassed by the method of the presentinvention.

A further aspect of the present invention relates to the use of theinvention in relation to the treatment and/or prophylaxis of diseaseconditions or other unwanted conditions. Without limiting the presentinvention to any one theory or mode of action, the regulation ofendothelial cell activity, and in particular intercellular vascularendothelial cell permeability, is an essential requirement in terms ofcontrolling the passage of solutes and cellular from the circulation tothe tissues both in terms of normal physiology and in the context ofmany unwanted pathologies. For example, newly formed micro-vessels andtumours are highly permeable, thereby facilitating the deposition offibrin in tumours which supports and promotes cell adhesion andmigration, essential steps in the angiogenic response. In anotherexample, chronic inflammatory states such as rheumatoid arthritis andatherosclerosis are characterised by vessel hyper-permeability whichallows increased transmigration of inflammatory cells across theactivated endothelium. A number of factors have been described whichpromote this endothelial cell leakiness, including the activity ofthrombin. Without limiting the present invention to any one theory ormode of action, it is thought that thrombin, for example, acts byinducing changes in junction or molecules such as PECAN-1 andVE-cadherin or their associated signalling molecules, such as thecateinins. Accordingly, the present invention is particularly useful,but in no way limited to, use as a therapy to down-regulateintercellular vascular endothelial cell permeability where an individualis suffering from an unwanted inflammatory condition or tumourdevelopment. Alternatively, the up-regulation of intercellular vascularendothelial cell permeability may be desirable where it is necessarythat passage of solutes or cells is facilitated from the circulationinto the tissue, such as for the purpose of facilitating angiogenesis intransplanted organs or limbs or in tissues which are subject to amyloidplaque deposition.

The present invention therefore contemplates a method for the treatmentand/or prophylaxis of a condition characterised by aberrant, unwanted orotherwise inappropriate endothelial cell activity in a mammal, saidmethod comprising modulating the functional activity of protein kinaseCζ wherein up-regulating protein kinase Cζ activity to a functionallyeffective level up-regulates said endothetial cell activity anddown-regulating protein kinase Cζ activity to a functionally ineffectivelevel down-regulates said cellular activity.

Preferably, said endothelial cells are vascular endothelial cells orlymphatic endothelial cells and said endothelial cell activity isintercellular permeability or intracellular permeability.

Reference to “aberrant, unwanted or otherwise inappropriate” endothelialcell functioning should be understood as a reference to under-activefunctioning, to physiologically normal functioning which isinappropriate in that it is unwanted or to over-active endothelial cellfunctioning. As detailed hereinbefore, there are a number of conditionswhich are dependent on the induction of the correct level of endothelialcell functioning, and in particular vascular endothelial cellfunctioning. For instance, and in relation to the preferred embodimentsdisclosed herein, in individuals experiencing an unwanted inflammatoryresponse, the down-regulation of protein kinase Cζ to a functionallyineffective level provides a means for this unwanted inflammatoryresponse to be retarded. In another example the development of new bloodvessels is a process which is central to the progress and/or continuanceof many disease conditions. For example, angiogenesis dependant diseasesinclude, but are not limited to, solid tumors, blood born tumors such asleulkemias, tumor metastasis, benign tumors, for example hemangiomas,angiofibromas, acoustic neuromas, neurofibroms, trachomas, and pyogenicgranulomas, rheumatoid arthritis, Crohn's disease, atherosclerosis,obesity, endometriosis, ocular angiogenic diseases, for example,diabetic retinopathy, retinopathy of prematurity (retrolentalfibroplasia), macular degeneration, corneal graft rejection, rubeosis,and neovascular glaucoma; psoriasis, facial and truncal telangiectasias,Osler-Webber Rendau syndrome (hereditary hemorrhagic telangiectasia).

In a most preferred embodiment, there is provided a method for thetreatment and/or prophylaxis of a condition characterised by unwantedendothelial cell activity in a mammal, said method comprisingadministering to said mammal an effective amount of an agent for a timeand under conditions sufficient to induce a functionally ineffectivelevel of protein kinase Cζ.

Preferably, endothelial cell is a vascular or lymphatic endothelial celland said activity is endothelial cell permeability. Most preferably,said condition is an inflammatory response or a solid tumour.

An “effective amount” means an amount necessary at least partly toattain the desired response, or to delay the onset or inhibitprogression or halt altogether, the onset or progression of theparticular condition being treated. The amount varies depending upon thehealth and physical condition of the individual to be treated, thetaxonomic group of the individual to be treated, the degree ofprotection desired, the formulation of the composition, the assessmentof the medical situation, and other relevant factors. It is expectedthat the amount will fall in a relatively broad range that can bedetermined through routine trials.

Reference herein to “treatment” and “prophylaxis” is to be considered inits broadest context. The term “treatment” does not necessarily implythat a subject is treated until total recovery. Similarly, “prophylaxis”does not necessarily mean that the subject will not eventually contracta disease condition. Accordingly, treatment and prophylaxis includeamelioration of the symptoms of a particular condition or preventing orotherwise reducing the risk of developing a particular condition. Theterm “prophylaxis” may be considered as reducing the severity or onsetof a particular condition. “Treatment” may also reduce the severity ofan existing condition.

The present invention further contemplates a combination of therapies,such as the administration of the modulatory agent together with otherproteinaceous or non-proteinaceous molecules which may facilitate thedesired therapeutic or prophylactic outcome.

Administration of molecules of the present invention hereinbeforedescribed [herein collectively referred to as “modulatory agent”], inthe form of a pharmaceutical composition, may be performed by anyconvenient means. The modulatory agent of the pharmaceutical compositionis contemplated to exhibit therapeutic activity when administered in anamount which depends on the particular case. The variation depends, forexample, on the human or animal and the modulatory agent chosen. A broadrange of doses may be applicable. Considering a patient, for example,from about 0.1 mg to about 1 mg of modulatory agent may be administeredper kilogram of body weight per day. Dosage regimes may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily, weekly, monthly or other suitable timeintervals or the dose may be proportionally reduced as indicated by theexigencies of the situation.

The modulatory agent may be administered in a convenient manner such asby the oral, intravenous (where water soluble), intraperitoneal,intramuscular, subcutaneous, intradermal or suppository routes orimplanting (e.g. using slow release molecules). The modulatory agent maybe administered in the form of pharmaceutically acceptable nontoxicsalts, such as acid addition salts or metal complexes, e.g. with zinc,iron or the like (which are considered as salts for purposes of thisapplication). Illustrative of such acid addition salts arehydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate,citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.If the active ingredient is to be administered in tablet form, thetablet may contain a binder such as tragacanth, corn starch or gelatin;a disintegrating agent, such as alginic acid; and a lubricant, such asmagnesium stearate.

Routes of administration include, but are not limited to,respiratorally, intratracheally, nasopharyngeally, intravenously,intraperitoneally, subcutaneously, intracranially, intradermally,intramuscularly, intraoccularly, intrathecally, intracereberally,intranasally, infusion, orally, rectally, via IV drip patch and implant.Preferably, said route of administration is oral.

In accordance with these methods, the agent defined in accordance withthe present invention may be coadministered with one or more othercompounds or molecules. By “coadministered” is meant simultaneousadministration in the same formulation or in two different formulationsvia the same or different routes or sequential administration by thesame or different routes. For example, the subject protein kinase Cζ maybe administered together with an agonistic agent in order to enhance itseffects. Alternatively, in the case of autoimmune inflanmmation, theprotein kinase Cζ antagonist may be administered together withimmunosuppressive drugs. By “sequential” administration is meant a timedifference of from seconds, minutes, hours or days between theadministration of the two types of molecules. These molecules may beadministered in any order.

Another aspect of the present invention relates to the use of an agentcapable of modulating the functionally effective level of protein kinaseCζ in the manufacture of a medicament for the regulation of endothelialcell activity in a mammal wherein up-regulating protein kinase Cζactivity to a functionally effective level up-regulates said endothelialcell activity and down-regulating protein kinase Cζ activity to afunctionally ineffective level down-regulates said endothelial cellactivity.

In another aspect the present invention relates to the use of proteinkinase Cζ or a nucleic acid encoding protein kinase Cζ in themanufacture of a medicament for the regulation of endothelial cellactivity wherein up-regulating protein kinase Cζ to a functionally levelup-regulates said endothelial cell activity.

According to these preferred embodiments, the subject endothelial cellsare preferably vascular or lymphatic endothelial cells. Still morepreferably the subject endothelial cell functioning is intercellular orintracellular endothelial cell permeability. Most preferably, saidfunctioning is down-regulated.

The term “mammal” and “subject” as used herein includes humans,primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys),laboratory test animals (eg. mice, rabbits, rats, guinea pigs),companion animals (eg. dogs, cats) and captive wild animals (eg. foxes,kangaroos, deer). Preferably, the mammal is human or a laboratory testanimal Even more preferably, the mammal is a human.

In yet another further aspect, the present invention contemplates apharmaceutical composition comprising the modulatory agent ashereinbefore defined and one or more pharmaceutically acceptablecarriers and/or diluents. Said agents are referred to as the activeingredients

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion or may be in the form of a cream or other formsuitable for topical application. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsuperfactants. The preventions of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the various sterilised active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When the active ingredients are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the active compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions in such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: a binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule encoding protein kinase Cζ or amodulatory agent as hereinbefore defined. The vector may, for example,be a viral vector.

The present invention is further defined by the following non-limitingExamples.

EXAMPLE 1 Angiopoietin-1 Inhibits Thrombin Induced Endothelial CellPermeability Changes by inhibition of Protien Kinase Cζ Materials andMethods

Reagents

Angiopoietin-1, obtained from Regeneron Inc was used as described(Gamble, J. R., Drew, J., Trezise, L., Underwood, A., Parsons, M.,Kasminkas, L., Rudge, J., Yancopoulos, G., Vadas, M. A. (2000) Circ Res.87:603-7; Papapetropoulos, A., Garcia-Cardena, G., Dengler, T. J.,Maisonpierre, P. C., Yancopoulos, G. D. and Sessa, W. C. (1999) LabInvest. 70:213-23). Bisindolylmaleimide I hydrochloride, Calphostin Cand LY294002 were from Calbiochem Novabiochem, (Croydon, Vic,Australia). Chelerythrine chloride and H-89 were from Biomol ResearchLaboratories, Inc (Plymouth Meeting, Pa., USA). Thrombin, proteaseinhibitor cocktail and FITC-conjugated dextran were from Sigma-Aldrich(St. Louis, Mo., U.S.A)

Cells and Cell Culture

Human umbilical vein endothelial cells were grown in M119 medium with20% foetal calf serum (FCS) as described (Litwin, M., Clark, K., Noack,L., Furze, J., Berndt, M., Albelda, S., Vadas, M., Gamble, J. (1997) JCell Biol. 139:219-28). Cells were used at passage 4 or less. Forinfection with adenoviral constructs, cells were grown to 80% confluenceand exposed to 2.5×10⁶ plaque forming units/25 cm² culture area for 2hours in M119 medium with 2% FCS and a further 22 hours with 20% FCS.

Recombinant Adenoviral Constructs

DNA constructs encoding FLAG-protein kinase Cζ, and FLAG-protein kinaseCζ T410A, in pCMV5, and Myr-protein kinase Cζ-FLAG in pCMV6 were agenerous gift from Dr. Alex Toker, Biomedical Research Institute,Boston, Mass., USA. Recombinant adenoviruses were constructed bysubcloning EcoR1 fragments from the pCMV5 constructs and HindIII/EcoRIfragment from the pCMV6 construct into the pAdEasy-1 vector (QbiogeneInc., Carlsbad, Calif., USA). Virus was amplified in HEK293 cells andpurified by CsC1 gradient ultracentifugation. Virus titres weredetermined using the TCID₅₀ method as recommended by Qbiogene.

Endothelial Permeability Assays

Assays were performed as described (Draijer et al. (1995) supra).Endothelial cells (10⁵) were cultured in transwells (3 μ-pore size,Corning Costar Corp, Cambridge, Mass., USA) for 24 hours in completemedium and then in 2% FCS medium for a further 24 hours. Cells werepre-treated with either bisindolylmaleimide I (10 nM or 6 μM),Calphostin C (100 nM), chelerythrine chloride (1 μM), H-89 (50 nM) orangiopoietin-1 (0.1 μg/ml), as required. FITC-conjugated dextran (0.2μg) was added to the upper chamber of all wells and cells were thentreated with thrombin (0.2 U/ml). The relative fluorescence in the lowerchambers of the transwells was determined using a LS 50B LuminescenceSpectrometer (Perkin Elmer, Beaconsfield, Buckinghamshire, UK;excitation wavelength, 485 nm; emission wavelength 530 nm) after 30minutes treatment. Adenovirus infected ECs were plated on to transwells24 hours after the above infection procedure and treated in the samemanner as uninfected cells.

Immunoblotting

Endothelial cells were plated into fibronectin coated flasks beforeinfection as above. Following infection, cells were serum depleted (0.2%FCS) overnight. Cells were then treated with angiopoietin-1, thrombinand/or bisindolylmaleimide I, as required, lysed in ice-cold lysisbuffer (50 mM Tris.HCl, pH 7.4, with 1% NP-40, 150 mM NaCl, 2 mM EGTA, 1mM NaPO4, 100 mM NaF, 10 mM sodium pyrophosphate and protease inhibitorcocktail). Protein concentrations were assayed using Bradford Reagent(BioRad, Hercules, Calif., USA). Equal amounts of protein were loadedonto 10% acrylamide gels, separated by SDS-PAGE, transferred to PVDFmembrane, blocked with 5% skim milk powder and 0.1% Triton-X100 inphosphate buffered saline, and probed with a polyclonal rabbit antibodydirected against phosphorylated Thr410 of the activation loop of proteinkinase Cζ (Chou, M. M., Hou, W., Johnson, J., Graham, L. K., Lee, M. H.,Chen, C. S., Newton, A. C., Schaffhausen, B. S., Toker, A. (1998) CurrBiol. 8:1069-77). This antibody was a generous gift from Dr Alex Toker.After washing, membranes were incubated with anti-rabbit secondaryantibody and reactive bands were detected by chemiluminescence (ECLWestern Blotting Detection Reagents, Amersham Pharmacia Biotech, LittleChalfont, England, UK). Membranes were stripped using stripping buffer(Re-Blot Plus Western Blot Recycling Kit, Chemicon, Temecula Calif.,USA) and re-probed with rabbit anti-protein kinase Cζ immunoaffinitypurified IgG (Upstate Biotechnology, Lake Placid N.Y., USA).

Immunofluorescence

6×10⁴ endothelial cells were cultured in fibronectin coated glass LabTekchamber slides (Nalge Nunc International, Naperville, Ill., USA) andincubated for 3 days prior to staining. Cells were then treated withangiopoietin-1, thrombin and/or bisindolylmaleimnide I, as required.Cells were washed once in PBS, fixed in 4% paraformnaldehyde/PBS for 5minutes, permeabilized with acetone for 5 min at −20° C. and then washedtwice with PBS. The fixed cells were incubated with rabbit anti-proteinkinase Cζ immunoaffmity purified IgG or anti-protein kinase Cλ (BDTransduction Laboratories, San Diego, Calif., USA) overnight at 4° C.followed by FITC-conjugated anti-rabbit antibody (Rockland,Gilbertsville Pa., USA). Coverslips were mounted using fluorescentmicroscopy mounting medium (Dako Corp., Carpinteria, Calif., USA). Cellswere imaged by epifluorescent microscopy on an Olympus BX-51 microscope(Olympus, Hamburg, Germany) equipped with excitation filters forfluorescein (494 nm) acquired to a Photometrics Cool Snap FXcharge-coupled device camera (Roper Scientific GmbH, Germany). Imageswere adjusted for brightness and contrast using V⁺⁺ software (DigitalOptics Ltd., Aucldand, New Zealand).

EXAMPLE 2 Thrombin-Induced Increased in EC Permeability are Inhibited byBlocking Protein Kinease Cζ Signalling

Thrombin signaling in vascular EC is mediated by the protease-activatedreceptor PAR-1 and protein kinase Cs are down-stream targets of PAR1(Coughlin, S. R. (2000) Nature 407:258-64). To clarify the role ofprotein kinase C and the specific isoforms in thrombin-inducedpermeability, we took advantage of the varying specificity of proteinIcinase C inhibitors. We used Chelerythrine Chloride, an inhibitor ofall protein Icinase C isoforms (Laudanna, C., Mochly-Rosen, D., Liron,T., Constantin, G., Butcher, E. C. (1998) J Biol Chem. 273:30306-15),Calphostin C, an inhibitor of classical and novel protein kinase Cs(Kobayashi, E., Nakano, H., Morimoto, M., Tamaoki, T. (1989) BiochemBiophys Res Commun. 159:548-53), and bisindolylmaleimide I, aconcentration dependent inhibitor of protein kinase C (Martiny-Baron,G., Kazanietz, M. G., Mischak, H., Blumberg, P. M., Kochs, G., Hug, H.,Marme, D., Schachtele, C. (1993) J Biol Chem. 268:9194-7), whereby at100 nM it inhibits classical and novel protein kinase C isoforms whileat 6 μM it inhibits all protein cinase C isoforms (Martiny-Baron et al.(1993) supra; Uberall, F., Hellbert, K., Kampfer, S., Maly, K.,Villunger, A., Spitaler, M., Mwanjewe, J., Baier-Bitterlich, G., Baier,G., Grunicke, H. H. (1999) J Cell Biol. 144:413-25). Using the passageof FITC labelled dextran through monolayers of human umbilical veinendothelial cells as a measure of permeability we found that bothbisindolylmaleimide I at 6 μM (FIG. 1 a) and chelerythrine chloride at 1μM (FIG. 1 b) inhibited thrombin stimulation of endothelial cellpermeability supporting the involvement of protein kinase C. Highconcentrations of bisindolylmaleimide I can also inhibit protein kinaseA (PKA), however, the specific protein kinase A inhibitor H-89 has noeffect on thrombin induced permeability changes (FIG. 1 c). Calphostin Cand bisindolylmaleimide I at 100 nM had no effect on thethrombin-induced increases of endothelial cell permeability (FIG. 1 d, 1a). Together the use of these inhibitors suggested that classical andnovel protein kinase Cs are not involved but atypical protein kinase Cisoforms are central to thrombin-induced endothelial cell permeabilityincreases.

The two atypical protein kinase C isoforms protein kinase Cζ and proteinkinase Cλ are present in endothelial cells (Li, H., Oehrlein, S. A.,Wallerath, T., Ihrig-Biedert, I., Wohlfart, P., UIlshofer, T., Jessen,T., Herget, T., Forstermann, U., Kleinert, H. (1998) Mol Pharmacol53:630-7). To determine if one or both of these isoforms are involved inthrombin-mediated permeability changes, confluent quiescent endothelialcells were stained with anti-protein kinase Cζ and anti-protein kinaseCλ antibodies. Both protein kinase Cζ and protein kinase Cλ weredistributed throughout the cytoplasm (FIG. 2 b, 2 e). When confluentmonolayers of endothelial cell were treated with thrombin, proteinkinase Cζ localized to the cell membrane (FIG. 2 c) while proteinIcinase Cλ remained evenly distributed despite significant contractionof individual cells, characteristic of the response to thrombinstimulation (FIG. 2 f). As targeting of protein kinase C isoforms to theplasma membrane indicates enzyme activation Nishizuka, Y. (2001) AlcoholClin Exp Res. 25:3S-7S), this observation is consistent with thrombinstimulation activating protein kinase Cζ but not protein kinase Cλ.

EXAMPLE 3 Activation of Protein Kinase Cζ Increases the Permeability ofEndothelial Cell

To confirm that protein kinase Cζ is a mediator of thrombin stimulatedincreases in endothelial cell permeability, wild-type, dominant-negativeand constitutively active protein kinase Cζ were over-expressed inendothelial cell by infection with adenovirus carrying these constructs.Dominant-negative protein kinase Cζ has the critical threonine of theactivation-loop, at position 410, mutated to alanine (Chou et al. (1998)supra). Constitutively active protein kinase Cζ results from fusion ofthe amino-terminal myristoylation sequence of p60 c-Src to the aminoterminus of protein kinase Cζ, thereby constitutively targeting theresultant protein to the cell membrane (Chou et al. (1998) supra). Cellsover-expressing wild-type protein kinase Cζ responded to thrombin in asimilar way to cells infected with empty vector (data not shown).Infection of endothelial cell with adenovirus carrying thedominant-negative protein kinase Cζ resulted in inhibition of thethrombin stimulated increase in endothelial cell permeability whencompared with cells infected with empty vector (FIG. 3 a) or wild-typeprotein kinase Cζ (data not shown). Conversely, endothelial cellsinfected with adenovirus carrying the constitutively active proteinkinase Cζ were highly permeable, even in the absence of thrombin, andthrombin stimulation had no further effect on permeability. Thus,protein Icinase Cζ appears to be critical in maintaining endothelialcell junction integrity and is implicated in thrombin inducedendothelial cell permeability increases.

EXAMPLE 4 Angiopoietin-1 Inhibits Thrombin-Induced Relocalisation andPhosphorylation of Protein Minase Cζ in Endothelial Cell

Angiopoietin-1 can inhibit the thrombin-induced increase of endothelialcell permeability (Gamble et al. (2000) supra) (FIG. 3 b). We thereforeinvestigated whether the angiopoietin-1 mediated inhibition was throughregulation of protein kinase Cζ. Staining of confluent quiescentendothelial cell with the anti-protein kinase Cζ antibody showed proteinkinase Cζ to be distributed throughout the cytoplasm (FIG. 2 b and FIG.4 a). Thrombin stimulation caused protein kinase Cζ to be localized tothe cell membrane (FIG. 2 c and FIG. 4 b). Concurrently, the shape ofthe endothelial cells changed with progressive retraction of themembrane and gaps between cells becoming evident. When endothelial cellswere pre-treated with angiopoietin-1, at 0.1 μg/ml for 30 minutes, orbisindolylmaleimide I, 6 μM for 15 minutes, followed by thrombintreatment (FIG. 4 c, 4 d), protein cinase Cζ localization to themembrane was dramatically decreased thus indicating that angiopoietin-1inhibition of thrombin stimulation may occur through inhibition ofprotein kinase Cζ translocation and activation.

Phosphorylation of the threonine in the activation-loop of proteinkinase Cs is the critical first step of PKC activation. This is rapidlyfollowed by autophosphorylation of a 30 threonine and a serine residuewithin the catalytic domain and concomitant translocation from thecytoplasm to the cell membrane (Parekh et al. (2000) supra; Chou et al.(1998) supra). Phosphorylated protein kinase CCζ could not be detectedusing an antibody specific for the phosphorylated Thr 410 within theactivation loop of protein kinase C following thrombin treatment ofnormal endothelial cells, presumably because the levels of the enzymeare low. Protein kinase Cζ was then over-expressed by adenoviralmediated infection. Thrombin treatment for 15 minutes increased thephosphorylation of over-expressed protein kinase Cζ (FIG. 5 a) whilepretreatment with angiopoietin-1 at 0.1 μg/ml for 30 minutes orbisindolylmaleimide I at 6 μM for 15 minutes inhibited thrombinstimulated protein kinase Cζ phosphorylation. As expected, endothelialcells infected with dominant-negative protein kinase Cζ showed noprotein Icinase Cζ phosphorylation in response to thrombin while thoseexpressing constitutively-active protein kinase Cζ showed constitutiveprotein kinase Cζ phosphorylation (FIG. 5 b).

Receptor-mediated activation of protein kinase Cζ may occur viaphosphatidylinositol-3-kinase (PI3-kinase) activation (Parekh et al.(2000) supra; Chou et al. (1998) supra; Le Good, J. A., Ziegler, W. H.,Parekh, D. B., Alessi, D. R., Cohen, P., Parker, P. J. (1998) Science281:2042-5). Indeed this appears to be the case in these cells aspre-treatment of endothelial cells with the PI3-kinase inhibitorLY294002 inhibited thrombin-induced permeability increases (FIG. 6) andblocked both protein kinase Cζ relocalisation (FIG. 4 e) andphosphorylation (FIG. 5 a) in response to thrombin. This is consistentwith thrombin activating a PI3-kinase-dependent pathway resulting inprotein kinase Cζ activation, leading to increases in endothelial cellpermeability.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

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1. A method of modulating endothelial cell activity, said methodcomprising modulating the functional activity of protein Cζ, whereinup-regulating protein kinase Cζ activity to a functionally effectivelevel up-regulates said cellular activity and down-regulating proteinkinase Cζ activity to a functionally ineffective level down-regulatessaid cellular activity.
 2. The method according to claim 1, wherein saidendothelial cell is a vascular endothelial cell or a lymphaticendothelial cell.
 3. The method according to claim 1, wherein saidcellular activity is endothelial cell permeability.
 4. The methodaccording to claim 3, wherein said endothelial cell permeability isintercellular or intracellular.
 5. The method according to claim 4,wherein said permeability is thrombin-induced vascular endothelial cellpermeability.
 6. The method according to claim 1, wherein saidmodulation is up-regulation of protein kinase Cζ activity and saidup-regulation is achieved by introducing into said endothelial cell anucleic acid molecule encoding protein kinase Cζ or functionalequivalent, derivative or homologue thereof or the protein kinase Cζexpression product or functional derivative, homologue, analogue,equivalent or mimetic thereof.
 7. The method according to claim 1,wherein said modulation is achieved by contacting said endothelial cellwith a proteinaceous or non-proteinaceous molecule which modulatestranscriptional and/or translational regulation of the protein kinase Cζgene.
 8. The method according to claim 1, wherein said modulation isup-regulation of protein kinase Cζ activity and said up-regulation isachieved by contacting said endothelial cell with a proteinaceous ornon-proteinaceous molecule which functions as an agonist of the proteinkinase Cζ expression product.
 9. The method according to claim 1,wherein said modulation is down-regulation of protein kinase Cζ activityand said down-regulation is achieved by contacting said endothelial cellwith a proteinaceous or non-proteinaceous molecule, which functions asan antagonist to the protein kinase Cζ expression product.
 10. Themethod according to claim 9, wherein said molecule is angiopoietin-1 orfunctional derivative, homologue, analogue, equivalent or mimeticthereof.
 11. The method according to claim 9, wherein said molecule ischelerythrine chloride or bisindoylmaleimide I or functional derivative,homologue, analogue, equivalent or mimetic thereof.
 12. The methodaccording to claim 9, wherein said molecule is a mutant protein kinaseCζ, which mutant is characterised by substitution of the threonineresidue at position 410 of the activation loop to alanine.
 13. Themethod according to claim 1, wherein said endothelial cell activity ismodulated in vivo.
 14. The method according to claim 13, wherein saidendothelial cell activity is modulated in vitro.
 15. A method ofregulating endothelial cell activity in a mammal, said method comprisingmodulating the functional activity of protein kinase Cζ in said mammalwherein up-regulating protein kinase Cζ activity to a functionallyeffective level up-regulates said endothelial cell activity anddown-regulating protein kinase Cζ activity to a functionally ineffectivelevel down-regulates said endothelial cell activity.
 16. The methodaccording to claim 15, wherein said endothelial cell is a vascularendothelial cell or a lymphatic endothelial cell.
 17. The methodaccording to claim 15, wherein said cellular activity is endothelialcell permeability.
 18. The method according to claim 17, wherein saidendothelial cell permeability is intercellular or intracellular.
 19. Themethod according to claim 18, wherein said permeability isthrombin-induced vascular endothelial cell permeability.
 20. The methodaccording to claim 15, wherein said modulation is up-regulation ofprotein kinase Cζ activity and said up-regulation is achieved byintroducing into said endothelial cell a nucleic acid molecule encodingprotein kinase Cζ or functional equivalent, derivative or homologuethereof or the protein kinase Cζ expression product or functionalderivative, homologue, analogue, equivalent or mimetic thereof.
 21. Themethod according to claim 15, wherein said modulation is achieved bycontacting said endothelial cell with a proteinaceous ornon-proteinaceous molecule which modulates transcriptional and/ortranslational regulation of the protein kinase Cζ gene.
 22. The methodaccording to claim 15, wherein said modulation is up-regulation ofprotein kinase Cζ activity and said up-regulation is achieved bycontacting said endothelial cell with a proteinaceous ornon-proteinaceous molecule which functions as an agonist of the proteinkinase Cζ expression product.
 23. The method according to claim 15,wherein said modulation is down-regulation of protein kinase Cζ activityand said down-regulation is achieved by contacting said endothelial cellwith a proteinaceous or non-proteinaceous molecule which functions as anantagonist to the protein kinase Cζ expression product.
 24. The methodaccording to claim 23, wherein said molecule is angiopoietin-1 orfunctional derivative, homologue, analogue, equivalent or mimeticthereof.
 25. The method according to claim 23, wherein said molecule ischelerythrine chloride or bisindoylmaleimide I or functional derivative,homologue, analogue, equivalent or mimetic thereof.
 26. The methodaccording to claim 23, wherein said molecule is a mutant protein kinaseCζ which mutant is characterised by substitution of the threonineresidue at position 410 of the activation loop to alanine.
 27. A methodfor the treatment and/or prophylaxis of a condition characterised byaberrant, unwanted or otherwise inappropriate endothelial cell activityin a mammal, said method comprising modulating the functional activityof protein kinase Cζ wherein up-regulating protein kinase Cζ activity toa functionally effective level up-regulates said endothelial cellactivity and down-regulating protein kinase Cζ activity to a functionalineffective level down-regulates said endothelial cell activity.
 28. Themethod according to claim 25, wherein said endothelial cell is avascular endothelial cell or lymphatic endothelial cell.
 29. The methodaccording to claim 25, wherein said cellular activity is endothelialcell permeability.
 30. The method according to claim 29, wherein saidendothelial cell permeability is intercellular or intracellular.
 31. Themethod according to claim 30, wherein said permeability isthrombin-induced vascular endothelial cell permeability.
 32. The methodaccording to claim 27, wherein said modulation is up-regulation ofprotein kinase Cζ activity and said up-regulation is achieved byintroducing to said mammal a nucleic acid molecule encoding proteinkinase Cζ or functional equivalent, derivative or homologue thereof orthe protein kinase Cζ expression product or functional derivative,homologue, analogue, equivalent or mimetic thereof.
 33. The methodaccording to claim 27, wherein said modulation is achieved byintroducing to said mammal a proteinaceous or non-proteinaceous moleculewhich modulates transcriptional and/or translational regulation of theprotein kinase Cζ gene.
 34. The method according to claim 27, whereinsaid modulation is up-regulation of protein kinase Cζ activity and saidup-regulation is achieved by introducing to said mammal a proteinaceousor non-proteinaceous molecule which functions as an agonist of theprotein kinase Cζ expression product.
 35. The method according to claim27, wherein said modulation is down-regulation of protein kinase Cζactivity and said down-regulation is achieved by introducing to saidmammal a proteinaceous or non-proteinaceous molecule which functions asan antagonist to the protein kinase Cζ expression product.
 36. Themethod according to claim 35, wherein said molecule is angiopoietin-1 orfunctional derivative, homologue, analogue, equivalent or mimeticthereof.
 37. The method according to claim 35, wherein said molecule ischelerythrine chloride or bisindoylmaleimide I or functional derivative,homologue, analogue, equivalent or mimetic thereof.
 38. The methodaccording to claim 35, wherein said molecule is a mutant protein kinaseCζ, which mutant is characterised by substitution of the threonineresidue at position 410 of the activation loop to alanine.
 39. Themethod according to claim 29, wherein said condition is an inflammatoryresponse.
 40. The method according to claim 29, wherein said conditionis unwanted angiogenesis.
 41. The method according to claim 40, whereinsaid condition is solid tumors, blood born tumors, tumor metastasis,benign tumors, rheumatoid arthritis, Crohn's disease, atherosclerosis,obesity, endometriosis, ocular angiogenic diseases, psoriasis, facialand truncal telangiectasias, or Osler-Webber Rendau syndrome. 42-48.(canceled)